Distinctive molecular features of regenerative stem cells in the damaged male germline

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Degust code for analysis of bulk RNA-Seq data is provided in a Zenodo repository (https://doi.org/10.5281/zenodo.3258932). Seurat for scRNA-Seq analysis is available from GitHub (https://github.com/satijalab/seurat/). SCENIC and scVelo are available from https:// scenic.aertslab.org/ and https://scvelo.org respectively.
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March 2021
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Life sciences study design
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Sample size
No statistical method was used to predetermine sample sizes. Randomization No specific randomization methods were used. Age matched wildtype male mice were randomly allocated into specific cages by animal staff prior to the experiment and cages were randomly assigned into individual experimental groups. Oct4-GFP mice were assigned to experiment in Figure 1f according to genotype but randomly allocated into experimental groups. For experiments using wildtype cultured spermatogonia, cells were maintained under the same conditions and randomly assigned to treatment groups.

Blinding
No blinding methods were used as specific procedures and treatments were performed and experimenters needed to be aware of group assignment.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. PE-Cy7-conjugated anti-MCAM clone ME-9F1 1:4000 Biolegend 134713, validation https://www.biolegend.com/en-us/products/pe-cyanine7-anti-mouse-cd146-antibody-9322. Statement: FC-Quality tested Goat polyclonal anti-uPAR 1:100 R&D AF534, validation https://www.rndsystems.com/products/mouse-upar-antibody_af534/. 293HEK cells (originally from ATCC) were used for production of lentivirus.

Authentication
No additional authentication of 293HEK line was performed.

Mycoplasma contamination
No additional testing for mycoplasma was performed.

Animals and other organisms
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Wild animals
This study did not use wild animals.
Field-collected samples This study did not use samples collected from the field.

Ethics oversight
Animal studies were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were subject to approval by the Monash University and Medical Centre Animal Ethics Committees (Projects MARP-2015-025 andMMCB-2020-15).
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Flow Cytometry
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Methodology Sample preparation
For analysis and sorting of live testis cells (PMID: 30126904): Decapsulated and minced adult testes were washed in phosphate-buffered saline (PBS) to remove spermatozoa and debris. A single cell suspension was then generated by digestion of tubules with 1mg/ml type II collagenase (Sigma) in un-supplemented DMEM (Thermo) with 40 U/ml DNase I at 37 degrees Celsius for 10 minutes with occasional agitation. Cells were dissociated in PBS with 2% fetal bovine serum (FBS), put through a 70 micron cell strainer and washed in PBS prior to use. Harvested cells were stained for 25 minutes on ice with antibodies in PBS with 2% FBS. For analysis of fixed and permeabilised testis cells (PMID: 28867346): Single cell suspensions were generated from washed testis tubules by sequential digest first in 1mg/ml type IV collagenase (Sigma) in un-supplemented DMEM (Thermo) with 40 U/ml DNase I at 37 degrees Celsius for 10 minutes with occasional agitation then in 0.25% Trypsin (Thermo) in PBS with 40 U/ ml DNase I at 37 degrees Celsius for 5 minutes . Cells were dissociated in PBS with 10% fetal bovine serum (FBS), put through a 70 micron cell strainer and washed prior to use. Harvested cells were stained for 30 minutes at room temperature with antibodies in PBS with 2% FBS.

Instrument
Cells were sorted with an Influx Cell Sorter (BD Biosciences) and analyzed using an LSR Fortessa X-20 (BD Biosciences).

Software
Data processed with FlowJo software v8.7 Cell population abundance For FACS sorting experiments, purity of isolated spermatogonia was approx. 95% as determined by single cell RNA-Seq analysis (Supplementary Table 7).

Gating strategy
For analysis and sorting, a FSC/SSC gate was used to exclude cell debris and clumps (approx. 70% cells retained). Doublets